Aptamer functionalized magnetic blade spraying device and small mass spectrum on-site rapid detection method

Abstract

The invention discloses an adapter functionalized magnetic blade spraying device which comprises a mass spectrometer (2) and a blade spraying ionization device which are connected through an alligator clip (1), the blade spraying ionization device comprises a blade (3) and a magnet (4) located below the blade (3), and the blade (3) comprises a detection solution. The detection solution comprises the aptamer functionalized magnetic nanoparticles and a sample. According to the device disclosed by the invention, aptamer functionalized magnetic nanoparticles, a blade spray ionization technology and a small portable mass spectrometer are ingeniously combined together. The invention further discloses a small portable mass spectrum on-site rapid detection method adopting the aptamer functionalized magnetic blade spraying device, specific recognition of a target object can be achieved through the aptamer functionalized magnetic nanoparticles, and the detection sensitivity is improved through nucleic acid dye mass spectrum labels adsorbed on the aptamers; the blade spray ionization technology and the small portable mass spectrometer can realize rapid, direct and real-time on-site analysis of the target object.

Description

Technical Field

[0001] The invention relates to the field of inspection and detection, in particular to an aptamer functionalized magnetic blade spray device and a small portable mass spectrometry on-site rapid inspection method using the device. Background Art

[0002] Coated blade spray is an efficient in situ ionization technology based on solid phase microextraction. It selectively enriches analytes from complex matrices by covering the tip of the blade with a coating, followed by desorption and ionization using a small amount of organic solvent. Immunomagnetic blade spray ionization technology replaces the surface coating of traditional coated blade spray with immunomagnetic microspheres. Its immune recognition combined with mass spectrometry significantly improves the selectivity of the method. In theory, by replacing the monoclonal antibodies on the surface of the magnetic beads, immune capture and analysis of a variety of food contaminants can be achieved. Despite the continuous advancement of immunoassay technology, the antibody development process is complex and time-consuming, so it is particularly necessary to find alternative recognition molecules. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an aptamer functionalized magnetic blade spray device with simple structure and easy operation and a small-scale mass spectrometry on-site rapid detection method using the device.

[0004] An aptamer functionalized magnetic blade spray device comprises a mass spectrometer and a blade spray ionization device connected via an alligator clip. The blade spray ionization device comprises a blade and a magnet located below the blade. A detection solution is included on the blade.

[0005] The aptamer-functionalized magnetic blade spray device of the present invention, wherein the detection solution comprises aptamer-functionalized magnetic nanoparticles and a sample.

[0006] The aptamer-functionalized magnetic blade spray device of the present invention, wherein the crocodile clip is made of copper, the aptamer-functionalized magnetic blade is fixed to the front end of the mass spectrometer injection port 5 mm by the crocodile clip, and the rear end of the crocodile clip is connected to the voltage of the mass spectrometer through a wire.

[0007] The aptamer-functionalized magnetic blade spray device of the present invention, wherein the mass spectrometer is a Miniβ small portable mass spectrometer.

[0008] A small portable mass spectrometry on-site rapid detection method using the aptamer-functionalized magnetic blade spray device is used to detect cell membrane glycoproteins in human blood samples. The method comprises the following steps:

[0009] (A) adding aptamer-functionalized magnetic nanoparticles and GelRed nucleic acid dye into a PCR tube, washing with deionized water after adsorption, adding deionized water and vortexing to obtain a resuspension of aptamer-functionalized magnetic nanoparticles;

[0010] (B) The nucleic acid dye-adsorbed aptamer-functionalized magnetic nanoparticles were resuspended and mixed with human serum samples, separated by a magnet, washed with deionized water, and vortexed with deionized water;

[0011] (C) The solution obtained in step (B) is added to the tip of the custom blade, and a magnet is placed underneath it to fix it. The blade is fixed to the front end of the mass spectrometer injection port with a copper alligator clip, and the rear end of the alligator clip is connected to the Miniβ small portable mass spectrometer through a wire;

[0012] (D) After absorbing the solution with a clean paper towel, methanol was added dropwise to the magnetic beads for mass spectrometry analysis.

[0013] The method of the present invention, wherein the parameters of the mass spectrometry detection analysis in step (D) are as follows: the spray voltage is 4.2 kV; the ionization mode is the positive ion mode; the collision gas is air; the collision time is 500 ms; the average number of times is 2 times; the continuous number of times is 3; the injection volume is 40 ms; and the mass-to-charge ratio cutoff at the low mass end of the injection is m / z 50.

[0014] The method of the present invention, wherein step (A) specifically comprises the following steps: adding 100 μL of aptamer-functionalized magnetic nanoparticles to 1 mL of 10X GelRed aqueous solution, adsorbing for 5 minutes; adsorbing the nanoparticles with a magnet, and washing with deionized water for 1 to 2 times; adding 100 μL of deionized water and vortexing to mix, to obtain a resuspension of aptamer-functionalized magnetic nanoparticles;

[0015] Step (B) specifically comprises the following steps: adjusting the pH value of the human serum sample to 6.0 using acetic acid; adding 10 μL of the aptamer-functionalized magnetic nanoparticle resuspension solution to 100 μL of the human serum sample, and incubating at room temperature for 30 minutes; adsorbing the nanoparticles with a magnet, and washing them with deionized water for 1 to 2 times; adding 20 μL of deionized water and vortexing to mix;

[0016] Step (C) specifically comprises the following steps: customizing a blade with a size of 4 cm×1.1 cm and a tip angle of 60°; dripping the solution obtained in step (B) onto the tip of the blade; placing a magnet of appropriate size at the bottom of the tip of the blade; fixing the blade to 5 mm from the front end of the mass spectrometer injection port with a copper alligator clip;

[0017] Step (D) specifically includes the following steps: absorbing the solution on the tip of the blade with a clean paper towel; dropping 10 μL of methanol on the surface of the magnetic beads and desorbing for 1 min; dropping 10 μL of a methanol solution containing 200 ng / mL crystal violet internal standard again; and applying voltage for spray ionization.

[0018] In the method of the present invention, the aptamer-functionalized magnetic nanoparticles are obtained by fixing the CD105 aptamer End-A2-T on hydrophilic polymer-modified magnetic nanoparticles to construct EBApt-pMNPs.

[0019] The method of the present invention, wherein the synthesis method of the aptamer-functionalized magnetic nanoparticles comprises the following steps:

[0020] FeCl3·6H2O and FeCl2·4H2O were dissolved in water, stirred for reaction, washed with water and ethanol, and dried to obtain Fe3O4 powder; Fe3O4, ethanol and 3-(trimethoxysilyl)propyl methacrylate were added to a flask, stirred for reaction, washed with water and ethanol, and dried to obtain Fe3O4@MPS powder; 4-vinyl-1,3-dioxolane-2-one (VEC) and hydroxyethyl acrylate (HEA) were selected as functional monomers, and azobisisobutyronitrile (AIBN) was used as initiator. Fe3O4@MPS, initiator AIBN, VEC, and HEA were added to a three-necked round-bottom flask containing 1,4-dioxane, stirred for reaction, and Fe3O4@MPS@p(VEC-AA) was obtained; finally, Fe3O4@MPS@p(VEC-AA) was added to the EBApt aqueous solution and stirred to obtain EBApt-pMNPs.

[0021] The method of the present invention, wherein the synthesis method of the aptamer functionalized magnetic nanoparticles specifically comprises the following steps: dissolving 5.4g FeCl3·6H2O and 2g FeCl2·4H2O in water, adjusting the pH value to 12.0 with NH3·H2O, stirring and reacting at 70°C for 3 hours, washing with water and ethanol, and drying to obtain Fe3O4 powder; adding 200mg Fe3O4, 100mL ethanol and 6mL 3-(trimethoxysilyl)propyl methacrylate into a flask, stirring and reacting at room temperature for 24 hours, washing with water and ethanol, and drying to obtain Fe3O4@MPS powder; selecting 4-vinyl-1,3-dioxolane-2-one and hydroxyethyl acrylate as functional monomers, AIBN as initiator, and adding 85mg Fe3O4@MPS, 6mg initiator AIBN, 265μL VEC and 48 μL HEA were added to a three-necked round-bottom flask containing 10 mL of the organic solvent 1,4-dioxane, and the reaction was stirred at 90 °C for 24 hours to obtain Fe3O4@MPS@p(VEC-AA); finally, 10 mg Fe3O4@MPS@p(VEC-AA) was added to 570 μL of 10 μM EBApt aqueous solution and stirred at room temperature for 24 hours to obtain EBApt-pMNPs.

[0022] The aptamer-functionalized magnetic blade spray device and detection method of the present invention are different from the prior art in that:

[0023] The aptamer-functionalized magnetic blade spray device of the present invention skillfully combines aptamer-functionalized magnetic nanoparticles, blade spray ionization technology and a small portable mass spectrometer. The aptamer-functionalized magnetic nanoparticles can realize the specific recognition of the target, the nucleic acid dye mass spectrometry label adsorbed on the aptamer improves the detection sensitivity, and the blade spray ionization technology and the small portable mass spectrometer can realize the rapid, direct and real-time on-site analysis of the target.

[0024] The combination of aptamer-functionalized magnetic nanoparticles and blade spray ionization technology in the method of the present invention is an efficient and powerful method that can give full play to the advantages of each, thereby providing a more powerful tool for the analytical field.

[0025] 1. The present invention uses aptamer-functionalized magnetic nanoparticles to specifically adsorb cell membrane glycoproteins in blood samples. The nucleic acid dye mass spectrometry labels adsorbed on the aptamers can improve the detection sensitivity and effectively reduce the matrix effect;

[0026] 2. The device and method of the present invention use a small portable mass spectrometer to eliminate the size and weight limitations of a large mass spectrometer, and also solve the complexity and time-consuming nature of the mass spectrometry method to a certain extent;

[0027] 3. The device of the present invention can realize online detection of different types of targets by replacing nucleic acid aptamers.

[0028] The aptamer-functionalized magnetic blade spray device and detection method of the present invention are further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the structure of a small portable mass spectrometry detection device for aptamer-functionalized magnetic blade spray in the present invention;

[0030] Figure 2 To determine the dye immobilization amount of aptamer-functionalized magnetic nanoparticles by UV spectrophotometry;

[0031] Figure 3 The small portable mass spectrometer secondary mass spectrometer is GelRed dye (A) and its internal standard crystal violet (B);

[0032] Figure 4 is a selectivity diagram for evaluating AGMP in the present invention;

[0033] Figure 5 The full scan primary mass spectrometer of cell membrane glycoproteins in blood samples detected by the device and method of the present invention; (A) 0 ng / mL and (B) 50 ng / mL cell membrane glycoproteins; (C) matrix matching calibration curve (blue) and standard solution calibration curve (red) of cell membrane proteins in the concentration range of 1 to 50 ng / mL;

[0034] The translations of all English words appearing in the drawings of the present invention are as follows:

[0035] Dye loading: dye loading;

[0036] CD105 Adsorption: CD105 protein adsorption;

[0037] Dye desorption: Dye desorption;

[0038] Aptamer: aptamer;

[0039] EBApt: Cell membrane glycoprotein-binding nucleic acid aptamer;

[0040] CD105: cell membrane glycoprotein;

[0041] CD26 / DPP4: dipeptidyl peptidase 4;

[0042] PD-L1: programmed death ligand 1;

[0043] HAS: human serum albumin;

[0044] AFP: alpha-fetoprotein;

[0045] Dye: Nucleic acid dye;

[0046] IS: internal standard;

[0047] Magnet: magnet;

[0048] EBApt-pMNPs: aptamer-functionalized hydrophilic polymer magnetic nanoparticles;

[0049] AGMP: aptamer-functionalized nucleic acid dye-loaded mass spectrometry probe;

[0050] Absorbance: absorption value;

[0051] Wavelength: wavelength;

[0052] Concentration: concentration;

[0053] pMNPs: hydrophilic polymer magnetic nanoparticles;

[0054] Relative Abundance: relative abundance;

[0055] Intensity ratio of m / z GelRed / IS: mass spectrometry peak intensity ratio of GelRed dye and internal standard;

[0056] Standard solution: Standard solution;

[0057] Matrix-matched: Matrix-matched. DETAILED DESCRIPTION

[0058] 1. Instruments and Devices

[0059] Miniβ small portable mass spectrometer (55cm×24cm×31cm, Beijing Qingpu Technology Co., Ltd.); Milli-Q ultrapure water device (Millipore, USA); pipette tips used in the experiment (Bio-Rad, USA).

[0060] The blade is made of ultra-thin rectangular iron sheet (length 20×width 10×thickness 0.3 mm, Shenzhen Youci Technology Co., Ltd.).

[0061] 2. Materials and Reagents

[0062] The cell membrane glycoproteins used in the experiment were purchased from Beijing Sino Biological Technology Co., Ltd.; GelRed nucleic acid dye was purchased from Beijing Sigma-Aldrich Company; crystal violet was from Beijing J&K Technology Co., Ltd.; human serum was purchased from Shanghai Braunschwei Biotechnology Co., Ltd. (Shanghai); all solutions were prepared with pure water prepared by the American Milli Q water purifier.

[0063] 3. Experimental setup

[0064] like Figure 1 As shown, an aptamer-functionalized magnetic blade spray device includes a mass spectrometer 2 and a blade spray ionization device connected by an alligator clip, the blade spray ionization device includes a blade 1 and a magnet 3 located below the blade 1, and a detection solution is included on the blade 1. The detection solution includes aptamer-functionalized magnetic nanoparticles and a sample, the alligator clip is made of copper, the blade 1 is fixed to the front end of the mass spectrometer 2 at 5 mm by the alligator clip, and the rear end of the alligator clip is connected to the voltage of the mass spectrometer 2 by a wire, and the mass spectrometer 2 is a Miniβ small portable mass spectrometer.

[0065] IV. Experimental Methods

[0066] The method for detecting cell membrane glycoprotein in human serum sample using the device of the present invention comprises the following steps:

[0067] (A) 100 μL of aptamer-functionalized magnetic nanoparticles were added to 1 mL of 10X GelRed aqueous solution and adsorbed for 5 min; the magnetic nanoparticles were adsorbed with a magnet and washed with deionized water for 1 to 2 times; 100 μL of deionized water was added and vortexed to obtain a resuspension of aptamer-functionalized magnetic nanoparticles.

[0068] (B) The pH value of human serum sample was adjusted to 6.0 using acetic acid; 10 μL of aptamer-functionalized magnetic nanoparticle resuspension was added to 100 μL of human serum sample and incubated at room temperature for 30 min; the magnetic nanoparticles were adsorbed by a magnet and washed 1 to 2 times with deionized water; 20 μL of deionized water was added and vortexed to mix.

[0069] (C) The solution obtained in step (B) is added to the tip of the custom blade, and a magnet is placed underneath it to fix it. It is then fixed to the front end of the mass spectrometer inlet with a copper crocodile clip, and the rear end of the crocodile clip is connected to the Miniβ small portable mass spectrometer through a wire; the size of the custom blade is 4 cm × 1.1 cm, and the tip angle is 60°.

[0070] (D) Use a clean paper towel to absorb the solution at the tip of the blade; add 10 μL of methanol to the surface of the magnetic beads and desorb for 1 min; add 10 μL of methanol solution containing 200 ng / mL crystal violet internal standard again; apply voltage for spray ionization.

[0071] The detection target is cell membrane glycoprotein, and the parameters of mass spectrometry detection and analysis are as follows: the spray voltage is 4.2 kV; the ionization mode is positive ion mode; the collision gas is air; the collision time is 500 ms; the average number is 2 times; the continuous number is 3; the injection volume is 40 ms; the mass-to-charge ratio cutoff of the low mass end of the injection is m / z 50.

[0072] 4. Results and Discussion

[0073] (1) Optimization of aptamer-functionalized magnetic nanoparticles

[0074] CD105 aptamer End-A2-T was immobilized on hydrophilic polymer-modified magnetic nanoparticles to construct EBApt-pMNPs. The amount of aptamer immobilized on the surface of EBApt-pMNPs was determined by measuring the UV absorption value of the supernatant before and after the immobilization process. The supernatants corresponding to EBApt-pMNPs-0 and EBApt-pMNPs-1 showed a higher level of aptamer, which was due to the low amount of HEA used in the synthesis process, which limited the low degree of polymerization on the surface of the material. With the increase of HEA dosage, the absorbance of the supernatant from EBApt-pMNPs-2 to EBApt-pMNPs-4 gradually decreased. Among them, EBApt-pMNPs-3 had the lowest absorbance, indicating that the amount of aptamer immobilized on its surface was the largest. The concentration of EBApt in the supernatant after the reaction was calculated to be 0.169 μM by the standard curve of aptamer concentration-UV absorption value. This result showed that 536 μL of 4.831 μM EBApt was successfully immobilized on 5 mg of pMNPs, that is, the immobilized amount of EBApt was 0.518 μmol / g.

[0075] Synthesis of aptamer-functionalized hydrophilic polymer-modified magnetic nanoparticles:

[0076] 5.4 g FeCl3·6H2O and 2 g FeCl2·4H2O were dissolved in water, and the pH value was adjusted to 12.0 with NH3·H2O, and then stirred at 70°C for 3 hours, washed with water and ethanol, and dried to obtain Fe3O4 powder. 200 mg Fe3O4, 100 mL ethanol, and 6 mL 3-(trimethoxysilyl)propyl methacrylate (MPS) were added to a flask, stirred at room temperature for 24 hours, washed with water and ethanol, and dried to obtain Fe3O4@MPS powder. 4-vinyl-1,3-dioxolane-2-one (VEC) and hydroxyethyl acrylate (HEA) were selected as functional monomers and AIBN as initiator. 85 mg Fe3O4@MPS, 6 mg initiator AIBN, 265 μL VEC, and 48 μL HEA were added to a three-necked round-bottom flask containing 10 mL of organic solvent 1,4-dioxane, and stirred at 90 °C for 24 hours to obtain Fe3O4@MPS@p(VEC-HEA). Finally, 10 mg Fe3O4@MPS@p(VEC-AA) was added to 570 μL 10 μM EBApt (aptamer corresponding to CD105 protein) aqueous solution and stirred at room temperature for 24 hours to obtain EBApt-pMNPs.

[0077] (2) Determination of the amount of nucleic acid dye fixed

[0078] EBApt-pMNPs were incubated with a sufficient amount of nucleic acid dye (GelRed) to construct an aptamer-functionalized nucleic acid dye-loaded mass spectrometry probe (AGMP). The loading amount of GelRed dye on AGMP is the key to the highly sensitive detection of CD105 protein. Figure 2 As shown, by plotting the standard curve of the peak absorption of GelRed at 280nm in the concentration range of 0.1 to 10X, the adsorption amount of GelRed on AGMP was measured to be 1.86X. It is worth noting that the UV absorption value of the magnetic polymer material without fixed aptamers is not much different from that of the initial dye, indicating that the magnetic nanoparticles themselves do not adsorb dye molecules.

[0079] (3) Optimization of adsorption conditions

[0080] The conditions affecting the sensitivity of AGMP were optimized, including solution pH, adsorption time, and adsorption temperature. First, the effect of solution pH on the adsorption efficiency of AGMP was studied. The experimental results showed that when the pH value was 6.0, the mass spectrometry signal intensity ratio of GelRed / IS reached 0.35, and as the pH value continued to increase or decrease to 9.0, the mass spectrometry signal intensity ratio gradually increased. Therefore, when the solution pH value was 6.0, the CD105 protein had the best effect in competing for binding to the aptamer. In addition, the incubation time of CD105 and AGMP was also investigated. After incubation with CD105, the mass spectrometry signal intensity ratio of the dye / internal standard decreased with time in the range of 0-30min and reached a peak at 30min, which was attributed to the high affinity between the aptamer and CD105. When the incubation time continued to increase, the mass spectrometry signal intensity ratio did not decrease further, indicating that a longer incubation time would not improve the sensitivity of the detection. Therefore, in subsequent experiments, the incubation time of AGMP and CD105 was selected as 30min. In addition, the incubation temperature of AGMP and CD105 was also optimized: when the incubation temperature of AGMP and CD105 was 4°C, the mass spectrometry signal intensity ratio of GelRed / IS was the largest, indicating that the CD105 protein had a poor ability to compete with the aptamer at this temperature. As the temperature increased, the mass spectrometry signal intensity ratio gradually decreased and reached a minimum at 37°C; as the temperature continued to increase, the mass spectrometry signal intensity ratio gradually increased. This may be due to the decrease in CD105 protein activity caused by the increase in temperature, resulting in a poor binding effect with the aptamer. In addition, since the mass spectrometry signal intensity ratios corresponding to 25°C and 37°C were not much different, a more convenient incubation temperature of 25°C was selected.

[0081] (4) Optimization of desorption conditions

[0082] The factors affecting the desorption of dyes were investigated. The desorption effects of water, methanol, acetonitrile, methanol-water (1:1, v / v) and acetonitrile-water (1:1, v / v) were compared. Methanol has the best desorption effect, which is better than acetonitrile and other solvents. This result shows that methanol can easily destroy the interaction between the aptamer and the dye molecules by inducing structural changes, thereby promoting the release of dye molecules. Subsequently, the effect of desorption time was investigated. The experimental results show that the dye molecules adsorbed on AGMP are rapidly released in a very short time (within 1 min), and the mass spectrometry signal intensity ratio of GelRed / IS does not increase significantly with the increase of desorption time. This result proves the high efficiency of the desorption process. Therefore, the optimal desorption conditions are: methanol as the desorption solvent and the desorption time is 1 min.

[0083] (5) Selective evaluation

[0084] Finally, the selectivity of AGMP was evaluated under the optimized conditions ( Figure 4 ). CD105, dipeptidyl peptidase 4 (CD26 / DPP4), programmed death ligand 1 (PD-L1), human serum albumin (HSA) and alpha-fetoprotein (AFP) were incubated with AGMP for 30 minutes and then desorbed using a methanol solution containing a crystal violet internal standard. Compared with the control group without protein incubation, the mass spectrometry signal intensity ratio of GelRed / IS in the elution solution corresponding to CD26 / DPP4, PD-L1 and HSA remained almost unchanged, indicating that there was almost no interaction between these proteins and the CD105 aptamer. The mass spectrometry signal intensity ratio corresponding to AFP decreased slightly, indicating that there was a weak interaction between it and the aptamer. It is worth noting that when incubated with CD105 protein, the MS intensity ratio decreased significantly, proving that there was a strong interaction between the CD105 protein and the aptamer. This result verifies the high specificity and affinity of CD105 protein binding to the aptamer.

[0085] (6) Methodological investigation

[0086] A series of CD105 protein solutions with a concentration range of 1 to 50 ng / mL were prepared using 200 ng / mL crystal violet as an internal standard for serum sample analysis. The mass spectrometry peak intensities of GelRed dye (m / z 412.22) and crystal violet (m / z 372.24) were compared. Figure 3 ) ratio and CD105 protein concentration ratio, and draw a calibration curve ( Figure 5 Red curve). According to 3σ / S and 10σ / S (σ is the standard deviation of 6 blank samples, S is the slope of the standard curve), the detection limit and quantification limit were calculated to be 0.2 ng / mL and 1.0 ng / mL, respectively. In order to evaluate the matrix effect of this method, the difference in the slope of the calibration curve between the matrix solution and the standard solution in the same concentration range was compared. Figure 5 As shown, compared with the standard solution calibration curve (red), the slope deviation of the matrix-matched calibration curve (blue) is 11.14%, indicating that this method can effectively reduce the influence of matrix effect.

[0087] First, the original content of CD105 in serum samples was determined by ELISA method: a standard curve was first established to determine the content of CD105 in three serum samples, which were 12.1 ng / mL, 6.7 ng / mL and 9.4 ng / mL, respectively. The results were of the same order of magnitude as those reported in the literature. Subsequently, different concentrations of CD105 (1 times the limit of quantification, 10 times the limit of quantification and 20 times the limit of quantification) were added to the serum samples and incubated with AGMP. The results were analyzed, and the spiked recoveries of the three samples were between 90.1% and 106.8%, and the relative standard deviations were between 3.6% and 5.1% (Table 2). In addition, compared with other reported CD105 detection methods, the method constructed by the present invention exhibited good linearity and considerable sensitivity (Table 1). These results demonstrate the feasibility of this method for the determination of CD105 in actual serum.

[0088] Table 1 Detection limit, quantification limit and linear relationship evaluation results of the method of the present invention

[0089]

[0090] Table 2 AGMP for the detection of spiked recovery of CD105 in human serum (n=6)

[0091]

[0092] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An aptamer-functionalized magnetic blade spray device, characterized in that: The invention comprises a mass spectrometer (2) and a blade spray ionization device connected by an alligator clip. The blade spray ionization device comprises a blade (1) and a magnet (3) located below the blade. A detection solution is disposed on the blade (1).

2. The aptamer-functionalized magnetic blade spray device according to claim 1, characterized in that: The detection solution comprises aptamer-functionalized magnetic nanoparticles and a sample.

3. The aptamer-functionalized magnetic blade spray device according to claim 2, characterized in that: The alligator clip (1) is made of copper. The blade (1) is fixed to the front end of the injection port of the mass spectrometer (2) by the alligator clip at 5 mm. The rear end of the alligator clip is connected to the voltage of the mass spectrometer (2) through a wire. The size of the blade (1) is 4 cm×1.1 cm, and the tip angle is 60°.

4. The aptamer-functionalized magnetic blade spray device according to claim 3, characterized in that: The mass spectrometer (2) is a Miniβ small portable mass spectrometer.

5. A small portable mass spectrometry on-site rapid detection method using the aptamer-functionalized magnetic blade spray device according to any one of claims 1 to 4, the method being used to detect cell membrane glycoproteins in human blood samples, characterized in that: The method comprises the following steps: (A) adding aptamer-functionalized magnetic nanoparticles and GelRed nucleic acid dye into a PCR tube, washing with deionized water after adsorption, adding deionized water and vortexing to obtain a resuspension of aptamer-functionalized magnetic nanoparticles; (B) The nucleic acid dye-adsorbed aptamer-functionalized magnetic nanoparticles were resuspended and mixed with human serum samples, separated by a magnet, washed with deionized water, and vortexed with deionized water; (C) The solution obtained in step (B) is added to the tip of the custom blade, and a magnet is placed underneath it to fix it. The blade is fixed to the front end of the mass spectrometer injection port with a copper alligator clip, and the rear end of the alligator clip is connected to the Miniβ small portable mass spectrometer through a wire; (D) After absorbing the solution with a clean paper towel, methanol was added dropwise to the magnetic beads for mass spectrometry analysis.

6. The method according to claim 5, characterized in that: The parameters of the mass spectrometry analysis in step (D) are as follows: the spray voltage is 4.2 kV; the ionization mode is the positive ion mode; the collision gas is air; the collision time is 500 ms; the average number is 2 times; the continuous number is 3; the injection volume is 40 ms; the mass-to-charge ratio of the injection low mass end cutoff is m / z 50.

7. The method according to claim 6, characterized in that: Step (A) specifically comprises the following steps: adding 100 μL of aptamer-functionalized magnetic nanoparticles to 1 mL of 10X GelRed aqueous solution, adsorbing for 5 minutes; adsorbing the nanoparticles with a magnet, and washing with deionized water for 1 to 2 times; adding 100 μL of deionized water and vortexing to obtain a resuspension of aptamer-functionalized magnetic nanoparticles; Step (B) specifically comprises the following steps: adjusting the pH value of the human serum sample to 6.0 using acetic acid; adding 10 μL of the aptamer-functionalized magnetic nanoparticle resuspension solution to 100 μL of the human serum sample, and incubating at room temperature for 30 minutes; adsorbing the nanoparticles with a magnet, and washing them with deionized water for 1 to 2 times; adding 20 μL of deionized water and vortexing to mix; Step (C) specifically comprises the following steps: customizing a blade with a size of 4 cm×1.1 cm and a tip angle of 60°; dripping the solution obtained in step (B) onto the tip of the blade; Place a magnet of appropriate size at the bottom of the blade tip; fix the blade to the mass spectrometer injection port 5 mm in front of the blade using a copper alligator clip; Step (D) specifically includes the following steps: absorbing the solution on the tip of the blade with a clean paper towel; dropping 10 μL of methanol on the surface of the magnetic beads and desorbing for 1 min; dropping 10 μL of a methanol solution containing 200 ng / mL crystal violet internal standard again; and applying voltage for spray ionization.

8. The method according to claim 5, characterized in that: The aptamer functionalized magnetic nanoparticles are prepared by fixing the CD105 aptamer End-A2-T on hydrophilic polymer modified magnetic nanoparticles to construct EBApt-pMNPs.

9. The method according to claim 8, characterized in that: The synthesis method of aptamer functionalized magnetic nanoparticles comprises the following steps: dissolving FeCl3·6H2O and FeCl2·4H2O in water, stirring for reaction, washing with water and ethanol, and drying to obtain Fe3O4 powder; adding Fe3O4, ethanol and 3-(trimethoxysilyl)propyl methacrylate into a flask, stirring for reaction, washing with water and ethanol, and drying to obtain Fe3O4@MPS powder; selecting 4-vinyl-1,3-dioxolane-2-one and hydroxyethyl acrylate as functional monomers, and azobisisobutyronitrile as initiator, adding Fe3O4@MPS, initiator AIBN, VEC, and HEA into a three-necked round-bottom flask containing 1,4-dioxane, stirring for reaction to obtain Fe3O4@MPS@p; finally, adding Fe3O4@MPS@p into an EBApt aqueous solution, stirring to obtain EBApt-pMNPs.

10. The method according to claim 9, characterized in that: The synthesis method of the aptamer functionalized magnetic nanoparticles specifically includes the following steps: dissolving 5.4g FeCl3·6H2O and 2g FeCl2·4H2O in water, adjusting the pH value to 12.0 with NH3·H2O, stirring and reacting at 70°C for 3 hours, washing with water and ethanol, and drying to obtain Fe3O4 powder; adding 200mg Fe3O4, 100mL ethanol and 6mL 3-(trimethoxysilyl)propyl methacrylate into a flask, stirring and reacting at room temperature for 24 hours, washing with water and ethanol, and drying to obtain Fe3O4@MPS powder; selecting 4-vinyl-1,3-dioxolane-2-one and hydroxyethyl acrylate as functional monomers, and azobisisobutyronitrile as initiator, and adding 85mg Fe3O4@MPS, 6mg initiator AIBN, 265μLVEC, and 48μL HEA was added into a three-necked round-bottom flask containing 10 mL of the organic solvent 1,4-dioxane, and the mixture was stirred at 90 °C for 24 h to obtain Fe3O4@MPS@p. Finally, 10 mg of Fe3O4@MPS@p was added into 570 μL of a 10.0 μM EBApt aqueous solution and stirred at room temperature for 24 h to obtain EBApt-pMNPs.

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